Studies on cancer have been intensively going on for more than 30 years. Nevertheless, incidence rate of cancer is still increasing and environmental pollution and unbalanced diet habit might be a reason. Every year, 10 million cancer patients are newly reported world-widely. WHO recognizes cancer the leading cause of death.
Even if new treatment methods for cancer have been developed, surgical operation, radio-therapy and chemotherapy are still co-treated largely. Generally, anticancer agent targets a specific cancer and does not have any effect on any other cancers. Besides, such anticancer agent can cause resistance. So, to increase effectiveness of cancer treatment, diverse novel anticancer agents have to be developed and a simple and easy screening method for them is also required.
While screening a target for a novel anticancer agent, the present inventors disclosed that invasion ability was increased in TMPRSS4 (transmembrane protease, serine 4) over-expressing cell line and when TMPRSS4 was inhibited by TMPRSS4 specific siRNA (small interfering RNA), invasion ability was reduced, suggesting that TMPRSS4 could be a target for the development of anticancer agent (Jung et al., Oncogene, 27: 2645-2647, 2008, Korean Patent Publication No 2007-0114970). The present inventors also confirmed in previous study that various factors were over-expressed when TMPRSS4 was over-expressed. Among those factors, the present inventors have focused on functions and roles of SIP1/ZEB2 and integrin alpha 5 (ITGA5).
SIP1/ZEB2 (Smad-interacting protein-1/Zinc finger E-box binding homeobox 2) is one of δEF-1 family which is two-handed Zinc finger nuclear factor. It is involved in epithelial mesenchymal transition (EMT) during developmental process. The mechanism is presumably as follows. SIP1/ZEB2 down-regulates the gene encoding a protein composing Gap-junction such as E-cadherin or desmosome (Vandevalle et al, Nucleic Acids Res. 33(20): 6566-6578, 2005). Besides, SIP1/ZEB2 accelerates cancer metastasis, in which double-negative feedback loop between SIP1/ZEB2 and microRNA-200 family is involved (Bracken et al., Cancer Res. 68(19):7846-54, 2008).
SIP1/ZEB2 and ZEB1, two members of δEF-1 family, have two zinc finger clusters. Each zinc finger cluster binds to E box (ex: CACCT). It was also confirmed by EMSA (electrophoretic mobility shift assay) that SIP1/ZEB2 binds to promoters of Xbra (Xenopus Brachyury), human integrin alpha 4 and human E-cadherin (Remacle et al., EMBO J. 18, 5073-5084, 1999). It is further known that SIP1/ZEB2 and ZEB1 are expressed in normal E-cadherin-negative tissues such as muscle cells and hematopoietic cells (Postigo and Dean, Proc. Natl. Acad. Sci. USA 97, 6391-6396, 2000). In particular, SIP1/ZEB2 binds to E-cadherin gene promoter to inhibit transcription of E-cadherin which is cell-cell adhesion molecule suppressing invasion, and accordingly cancer cell invasion is induced (Comijin et al., Mol. Cell 7: 1267-1278, 2001). In addition to SIP1/ZEB2, SNAI1 (snail homolog 1), SNAI2, TWIST and E47 are known as factors inducing cancer cell invasion and metastasis by suppressing E-cadherin transcription and inducing EMT (Peinado et al., Nature Rev. Cancer 7: 415-428, 2007). Those factors demonstrate different expression patterns according to the kind of cancer (Peinado et al., Nature Rev. Cancer 7: 415-428, 2007). SIP1/ZEB2 is largely expressed in ovarian cancer, stomach cancer, pancreatic cancer and squamous cell carcinoma (SCC), and ZEB1 is expressed in colon cancer and uterine cancer. TWIST and SNAI1 are up-regulated in diffuse subtype of stomach cancer, while SIP1/ZEB2 are up-regulated in intestinal subtype of stomach cancer (Rosivatz et al., Am. J. Pathol. 161: 1881-1891, 2002). The above results indicate that EMT and transition inducing factors make different combinations according to the type of cancer and thus play different roles. However, expression and functions of SIP1/ZEB2 in colon cancer have not been disclosed, yet.
ITGA5 (integrin subunit alpha5) is one of integrin subunits. Integrins are heterodimers comprising alpha and beta chains. After translation in extracellular domain, it forms light and heavy chains linked by disulfide bond and then binds to beta 1 to form a fibronectin receptor. Integrin family contains at least 25 combinations made by pairing of 18 alpha chains and 8 beta chains (van der Flier and Sonnenberg, Cell Tissue Res. 305:285-298, 2001). Integrin is a receptor mainly binding to extracellular matrix and is connected to intracellular actin cytoskeleton. Integrin is also known to be involved in cell-surface mediated signal transduction. Integrin alpha 5 beta 1 binds to fibronectin. Integrin alpha 5 is involved in progress, invasion and metastasis of liver cancer (Yao et al., Zhonghua Yi Xue Za Zhi. 77(5):382-384, 1997) and melanoma (Mortarini et al., Cancer Res. 52(16):4499-4506, 1992). Integrin alpha 5 beta 1 is highly expressed rather in strong carcinogenic colon cancer cell line than in weak carcinogenic colon cancer cell line (Gong et al., Cell Growth & Differentiation 8:83-90, 1997). In the meantime, integrin alpha 5 beta 1 expression is almost inhibited during colon cancer development and when integrin alpha 5 beta1 is re-expressed in the colon cancer cell line originally not-expressing integrin alpha 5 beta1, HER-2 is down-regulated by integrin alpha 5 beta1, suggesting that integrin alpha 5 beta 1 (that is integrin alpha 5) has tumor suppressor-like activity (Kuwada and Li, Mol. Biol. Cell 11:2485-2496, 2000; Kuwada et al., J. Biol. Chem. 280:19027-19035, 2005). However, it is still unknown what kind of role integrin alpha 5 (ITGA5) plays in colon cancer.
While screening a target material for screening anticancer agent or SIP1/ZEB2 inhibitor, the present inventors confirmed that cancer cell invasion was increased by SIP1/ZEB2 and integrin alpha 5 and when SIP1/ZEB2 was over-expressed, integrin alpha 5 was up-regulated. And further the present inventors completed this invention by developing a method for screening anticancer agent or SIP1/ZEB2 inhibitor using the said integrin alpha 5.